1. Field of the Invention
This invention relates to a method for culturing Bacillus subtilis, the cultured microorganism obtained by the method, a water-soluble vitamin K derivative originating in the cultured microorganism, and a food product, beverage, or feed containing the cultured microorganism or the water-soluble vitamin K derivative. More particularly, this invention relates to a method for culturing Bacillus subtilis in such a manner as to induce storage of a vitamin K derivative, particularly a menaquinone-7 (vitamin K2) derivative, in the largest amount within the cells of the Bacillus subtilis, a cultured product of Bacillus subtilis having a vitamin K derivative, particularly a menaquinone-7 (vitamin K2) derivative, accumulated in a large amount within the cells thereof in consequence of the culture by the method mentioned above, a water-soluble vitamin K derivative, particularly a water-soluble menaquinone-7 (vitamin K2) derivative, originating in the cultured product mentioned above, and a food product, beverage, or feed containing the cultured product or the water-soluble vitamin K derivative, particularly the water-soluble menaquinone-7 (vitamin K2) derivative.
2. Description of the Related Art
Vitamin K has been heretofore known as a factor found necessary during the coagulation of blood. Since the deficiency of this vitamin brings deterioration of the ability to coagulate blood, vitamin K is held to constitute itself one species of fat-soluble vitamin otherwise called an antihemorrhagic vitamin. As regards the cause for the deterioration of ability to coagulate blood thus brought about by the deficiency in this vitamin K, it has been suggested in recent years that vitamin K is indispensable for the biosynthesis of several blood coagulation factors including prothrombin. The amount of the vitamin K which is found necessary for the purpose of preventing the deterioration of the blood-coagulating ability is extremely small, i.e. on the order of xcexcg. Generally, since adults are supplied with this vitamin K by enterobacteria, they rarely contract the disease of vitamin K deficiency. The synthetic vitamins K1 and K2 are used as medicines for the therapy of the disease of the vitamin K deficiency hemorrhage. To date, the vitamin K has not attracted much attention because the concentrate of natural vitamin K1 has been utilized heretofore in the form of a food product for the purpose of preventing this disease.
It has been demonstrated in recent years, however, that vitamin K has a function of promoting ossification and a function of repressing bone resorption and that the density of bone is increased by the administration of the vitamin K. The osteoporosis is the pathosis of embrittlement of bone caused by senility or illness. Since this disease accompanies fracture and severe pain, it has been posing a serious social issue from the standpoint of geriatric therapy. A study conducted on patients with osteoporosis to determine their vitamin K levels in blood has demonstrated that the vitamin K levels average about one half of those of healthy persons. As a prospective medicine for the treatment of osteoporosis, therefore, the synthetic vitamin K is now under a clinical test. It has been established by the clinical test that in the treatment and prevention of osteoporosis, unlike the case of treating and preventing hemorrhage, the administration of vitamin K in such a large daily dose of not less than 45 mg is effective in increasing the amount of bone. The osteoporosis favors the prevention thereof rather than the treatment to be effected after the onset of the disease. For the sake of this prevention, daily ingestion of the vitamin K from food is commendable. The ingestion of the vitamin K in the dose mentioned above from the existing food seems to be difficult, however, because the question how much of the vitamin K ought to be ingested daily to increase the amount of bone and attain the prevention of osteoporosis remains yet to be elucidated.
The ingestion of vitamin K is preferred to be attained by the daily food as described above. In fact, vitamin K1 can be taken from greenish yellow vegetables and marine plants and vitamin K2 from such fermented food products as fermented soybeans (natto). An effort to attain the ingestion of 45 mg of the vitamin K, i.e. an amount reported to be effective in improving osteoporosis, from the commercially available food product, however, actually turns out to be a very difficult task as aptly evinced by the fact that a food product containing 1 ppm of vitamin K, for example, ought to be consumed in such a large amount as 45 kg daily to fulfill the ingestion under discussion. It is natto, among other food products, that has the largest vitamin K content on the order of ten-odd ppm. Even the natto so renowned ought to be consumed in such a large amount as some hundreds of g to several kg daily to fulfill the ingestion. From the standpoint of taste, it is difficult for any person to form the habit of eating the natto in such a large amount as mentioned above daily. In addition to this difficulty, the ingested vitamin K has a short half-life period. The vitamin K has such unsolved problems as failing to manifest the effect thereof fully satisfactory when it is orally ingested independently and tending to entail adverse reactions when it is ingested in an unduly large amount all at once. While the ingestion of the vitamin K in the concentrated form is commendable, the commercially available natural vitamin K concentrate which additionally incorporates therein prepared milk powder for the sake of preventing hemorrhage is expensive and the synthetic vitamin K offered as a pharmaceutical preparation is not usable as food.
Incidentally, in the class of vitamin K""s, it are only the vitamin K1 and K2 groups that occur in nature. The vitamin K1 is copiously contained particularly in green vegetables, vegetable oils, and marine products among other kinds of food. Seaweed, laver, and tea leaves, for example, contain some tens of ppm of vitamin K1 and soybean oil, spinach, and broccoli, for example, contain several ppm of vitamin K1. This vitamin K1 is synthesized by condensing 2-methyl-1,4-naphthoquinone and phytyl acetate. Further, the vitamin K2 group is known as varying homologs of menaquinone-1 to -14 (MK-1 to MK-14), depending on the difference in chain length. Among other homologs, particularly the menaquinone-7 (occasionally referred to simply as xe2x80x9cMK-7xe2x80x9d in the present specification) is a typical substance of the vitamin K2 and is synthesized mainly by Bacillus subtilis natto. In nature, the MK-7 is isolated only with unusual difficulty because it occurs in a relatively minute amount in the range of several to ten-odd ppm even in natto and has a short half-life period. So far, the invention of JP-A-08-73,396 has been known as the sole case of succeeding in preparing a lipid having a high MK-7 content.
Thus, quantity production of vitamin K2 by the use of such a microorganism as Bacillus subtilis natto has been attempted. Many studies have been known to have perfected methods for producing natural vitamin K2. Methods for collecting vitamin K2 from the culture broth of a microorganism belonging to genus Flavobacterium (JP-B-07-28,748 and JP-B-07-51,070) and methods for producing vitamin K by inoculating Bacillus subtilis natto to soy beam soup stock or soy-bean cake lees and fermenting the microorganism in the medium (JP-A-10-295,393, JP-A-08-19,378, JP-A-08-9,916, and JP-A-08-173,078) may be cited as examples of the outcomes of such studies. In addition to these methods, a method for obtaining a concentrated lipid containing natural vitamin K2, particularly natural MK-7, in a large amount by subjecting the fermented cells of Bacillus subtilis natto to extraction with such an organic solvent as alcohol, ether, ester, or ketone has been proposed (JP-A-08-73,396). The methods which use such a vitamin K-producing microorganism as Flavobacterium have the problem that the vitamin K2 obtained thereby can not be utilized in its unmodified form for a food product because the safety of Flavobacterium as food has not been established. Though the methods which prepare the vitamin K by the use of Bacillus subtilis natto indeed obtain cultures with relatively high vitamin K contents reaching the maximum of about 40 mg/litter of culture broth, the products thereof find utility only in heavily restricted applications because they are not water-soluble but fat-soluble bulks of vitamin K. Further, the lipid with a high natural menaquinone-7 content prepared by extracting the fermented cells of Bacillus subtilis natto with an organic solvent, despite the use of such raw materials as soy beans which are available for food, uses the organic solvent and, therefore, requires thorough removal of the organic solvent before it is used in food. This removal of the organic solvent necessitates provision of a special device intended exclusively therefor and entails an addition to the time required for the operation. The lipid of high MK-7 content to be obtained is a fat-soluble product, similarly to the products mentioned above, as clearly inferred from the designation thereof and, consequently, finds utility only in limited applications.
Many methods for producing vitamin K and MK-7 by using such microorganisms as Bacillus subtilis natto from the fermented broths of natto and such by-products as lees generated during the course of manufacture of natto and soup stock (namely for extracting vitamin K and MK-7 from the cells of the microorganism) have been reported in literature. Virtually no reports have been heretofore made concerning vitamin K and MK-7 stored within the cells of species of Bacillus subtilis represented by Bacillus subtilis natto.
An object of this invention, therefore, is to provide a method for culturing Bacillus subtilis in such a manner as to induce storage of a vitamin K derivative, particularly a menaquinone-7 (vitamin K2) derivative, in the largest amount within the cells of the Bacillus subtilis, a cultured product of Bacillus subtilis having a vitamin K derivative, particularly a menaquinone-7 (vitamin K2) derivative, accumulated in a large amount within the cells thereof in consequence of the culture by the method mentioned above, a water-soluble vitamin K derivative, particularly a water-soluble menaquinone-7 (vitamin K2) derivative, originating in the cultured product mentioned above, and a food product, beverage, or feed containing the cultured product mentioned above or the water-soluble vitamin K derivative, particularly the water-soluble menaquinone-7 (vitamin K2) derivative.
Another object of this invention, directed at rendering natural vitamin K, particularly natural MK-7, which either cannot be ingested at all or may be ingested only with difficulty, in a sufficient amount from the ordinary food product ingestible easily and daily, is to provide a method for culturing Bacillus subtilis in such a manner as to induce storage of a vitamin K derivative, particularly a menaquinone-7 (vitamin K2) derivative, in the largest amount within the cells of the Bacillus subtilis, a cultured product of Bacillus subtilis having a vitamin K derivative, particularly a menaquinone-7 (vitamin K2) derivative, accumulated in a large amount within the cells thereof in consequence of the culture by the method mentioned above, a water-soluble vitamin K derivative, particularly a water-soluble menaquinone-7 (vitamin K2) derivative, originating in the cultured product mentioned above, and a food product, beverage, or feed containing the cultured product mentioned above or the water-soluble vitamin K derivative, particularly the water-soluble menaquinone-7 (vitamin K2) derivative.
Still another object of this invention, directed at rendering natural vitamin K, particularly natural MK-7, which either cannot be ingested at all or may be ingested only with difficulty, in a sufficient amount from the ordinary food product ingestible easily and daily, is to provide a method for culturing Bacillus subtilis in such a manner as to induce storage of a vitamin K derivative, particularly a menaquinone-7 (vitamin K2) derivative, in the largest amount within the cells of the Bacillus subtilis, a cultured product of Bacillus subtilis having a vitamin K derivative, particularly a menaquinone-7 (vitamin K2) derivative, having effects of maintaining the level thereof in blood as heightened to a necessary level for a long time and at the same time excelling in safety accumulated in a large amount within the cells thereof in consequence of the culture by the method mentioned above, a water-soluble vitamin K derivative, particularly a water-soluble menaquinone-7 (vitamin K2) derivative, originating in the cultured product mentioned above, and a food product, beverage, or feed containing the cultured product mentioned above or the water-soluble vitamin K derivative, particularly the water-soluble menaquinone-7 (vitamin K2) derivative.
The present inventor, in appreciation of the true state of prior art mentioned above, has pursued a diligent study on the components of natto (Experientia, 43:1110, 1987; Acta Haematol., 84: 139, 1990; Fibrinolysis, 6: 86, 1992; Journal of Japan Society of Pharmacists, 30: 73, 1994; Bio-Industry, 14: 47, 1996) or the analysis of vitamin K in Bacillus subtilis natto and in blood (Japan Thrombotic Hemostasis Journal, 8: 287, 1997; Fibrinolysis and Proteolysis, Vol. 12, Supplement 1, 205, p. 75, 1998) and has consequently found that rather than the ingestion of the fermented product of Bacillus subtilis natto such as fermented soybeans or the vitamin K contained therein, the ingestion of live Bacillus subtilis itself is recognized to be highly effective in promoting the level of vitamin K, particularly MK-7, in plasma and that particularly the ingestion of Bacillus subtilis natto manifests effects of maintaining the level in plasma at an extremely high level as compared with the ingestion of other substances.
The present inventor has also found that Bacillus subtilis natto cultured to a specific stage of growth has MK-7 accumulated in a large amount in the cells thereof, that by ingesting the Bacillus subtilis natto or the fermented product thereof recovered at this stage, it is recognized to heighten the level of vitamin K, particularly MK-7, in plasma, and that the level of the vitamin K in plasma obtained in consequence of the ingestion is maintained for a long time. In addition to this knowledge, the inventor has also found that the vitamin K, particularly the MK-7, accumulated in the cells of Bacillus subtilis natto recovered at the stage mentioned above is soluble in water and has come to entertain expectations that applications of the water-soluble vitamin K, particularly MK-7, will greatly expand.
This invention has been perfected based on the knowledge as described above.
Specifically, the objects mentioned above can be accomplished by a method for culturing Bacillus subtilis, comprising the steps of culturing Bacillus subtilis and recovering the cells of said Bacillus subtilis before the vitamin K produced in the cells is released from the cells.
The objects mentioned above can be further accomplished by a cultured product of Bacillus subtilis obtained by the culturing method of this invention, a water-soluble vitamin K derivative originating in the cultured product of Bacillus subtilis, and a food product, beverage, or feed containing the fermented product mentioned above and/or the water-soluble vitamin K derivative.
The method for culturing Bacillus subtilis according to this invention is characterized by culturing Bacillus subtilis and recovering the cells of the Bacillus subtilis before the vitamin K produced in the cells is released from the cells. By the method of this invention, therefore, the Bacillus subtilis used therein can be recovered in a state having the vitamin K derivative, particular the menaquinone-7 derivative, stored in the largest amount within the cells thereof. For this reason, the water-soluble vitamin K, particularly the menaquinone-7 derivative, can be recovered in a larger amount from the cultured product as compared with the conventional method. Further, particularly when Bacillus subtilis natto is used, since the safety thereof has been ensured, the cultured product obtained by the method mentioned above or the food product, beverage, or feed containing the water-soluble vitamin K derivative originating in the cultured product contains the water-soluble vitamin K derivative, particularly the menaquinone-7 derivative, in a larger amount and possesses higher safety as compared with the conventional product. The practice of eating this food product, beverage, or feed can be expected to ensure efficiently simple daily ingestion of vitamin K and advance further the improvement of osteoporosis.
The water-soluble vitamin K derivative originating in the cultured product obtained by the method of the present invention embraces an epochal invention purporting that the vitamin K heretofore obtained in a fat-soluble form can be converted into a water-soluble quality. Further, the water-soluble vitamin K derivative has excellent photostability. This particular invention can be expected not only to enlarge appreciably the use found for the new vitamin K as compared with the conventional fat-soluble vitamin K but also to confer a very high value on this invention from the industrial point of view.
Further, since the practice of ingesting the food product, beverage, or feed containing the cultured product or the water-soluble vitamin K derivative can be expected to allow more effective prevention of osteoporosis because it permits the level of vitamin K in plasma to be efficiently heightened and enables the heightened level in plasma to last for a far longer period than the vitamin K assimilated from conventional medicines or food products.
The above and other objects, features and advantages of the present invention will become clear from the following description of the preferred embodiments.